Matrix effect in F2-isoprostanes quantification by HPLC–MS/MS: A validated method for analysis of iPF2α-III and iPF2α-VI in human urine

• We developed a HPLC–MS/MS method for the quantification of urinary F2-isoprostanes.
• A careful assessment of matrix effect and an accurate validation were carried out.
• We highlighted the importance of assessing matrix effect during method development.
• Ion suppression, due to the matrix, was reduced through optimization of the method.
• The assay was suitable for an accurate determination of endogenous F2-isoprostanes.

Liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) has become the method of choice for analysis in biological matrices, because of its high specificity and sensitivity. However, it should be taken into account that the presence of matrix components coeluting with analytes might interfere with the ionization process and affect the accuracy and precision of the assay. For this reason, the presence of a “matrix effect” should always be evaluated during method development, above all in complex matrix such as urine. In the present work, a HPLC–MS/MS method was developed for the quantification of urinary iPF2α-III and iPF2α-VI. A careful assessment of matrix effect and an accurate validation were carried out, in order to verify the reliability of quantitative data obtained. Ion suppression, due to the matrix components, was reduced through optimization of both chromatographic method and sample extraction procedure. Urine samples were purified by solid phase extraction (SPE) and the extracts injected into the HPLC–MS/MS system, equipped with a TurboIonSpray ionization source operated in negative ion mode (ESI−). Stable isotope-labeled analogues (iPF2α-III-d4 and iPF2α-VI-d4) were used as internal standards, and quantification was performed in multiple reaction monitoring (MRM) mode by monitoring the following mass transitions: m/z 353.4 → 193.2 for iPF2α-III, m/z 357.2 → 197.0 for iPF2α-III-d4, m/z 353.4 → 115.1 for iPF2α-VI, and m/z 357.4 → 115.1 for iPF2α-VI-d4. The validated assay, applied to the analysis of urinary samples coming from healthy and overweight subjects, resulted suitable for an accurate quantification of iPF2α-III and iPF2α-VI in human urine.